Cytotoxic Polyketides from a Marine-derived Fungus Aspergillus glaucus

J. Nat. Prod. 2008, 71, 1837–1842

1837

Cytotoxic Polyketides from a Marine-derived Fungus Aspergillus glaucus Lin Du, Tianjiao Zhu, Hongbing Liu, Yuchun Fang, Weiming Zhu, and Qianqun Gu* Key Laboratory of Marine Drugs, Chinese Ministry of Education, School of Medicine and Pharmacy, Ocean UniVersity of China, Qingdao 266003, People’s Republic of China ReceiVed May 20, 2008

Eight new aromatic polyketides (2, 4-6, 8, 14, 16, and 17) together with eight known analogues (3, 7, 9-13, and 15) were isolated from the marine-derived fungus Aspergillus glaucus. The structures and stereochemistry of the new compounds were elucidated by spectroscopic and chemical methods, and their cytotoxicities were evaluated against the HL-60 and A-549 cell lines. Fungal polyketides constitute a large family of secondary metabolites endowed with a high degree of structural diversity and various biological activities.1 These compounds include toxins such as aflatoxin B1 produced by Aspergillus species, psychoactive compounds such as xenovulene, and pharmaceuticals such as the cholesterol-lowering drug lovastatin.2 We have previously reported aspergiolide A (1), a novel cytotoxic anthraquinone derivative with a naphtho[1,2,3-de]chromene-2,7-dione skeleton, from cultures of the marine-derived fungus Aspergillus glaucus.7 The A. glaucus group is well known for producing aromatic polyketide mycotoxins,3 such as emodin, erythroglaucin, questin, physcion, physcion9-anthrone, catenarin, rubrocristin,4 physcion dianthranol, erythroglaucin,5 kotanin, and desmethylkotanin.6 In an effort to obtain more insight into the biosynthetic mechanisms and structure-activity relationships in this family of metabolites, our search for aromatic polyketides from this strain led to the discovery of eight new aromatic polyketides [namely, a new aspergiolide A analogue, aspergiolide B (2); three naphthyl ribofuranosides, isotorachrysone6-O-R-D-ribofuranoside (4), 8-methoxy-3-methyl-1-naphthalenol6-O-R-D-ribofuranoside (5), and 8-methoxy-1-naphthalenol-6-OR-D-ribofuranoside (6); two anthracene derivatives, isoasperflavin (8) and (+)-variecolorquinones A (14); and two bianthrones, (trans)and (cis)-emodin-physcion bianthrone (16 and 17)] and eight known analogues [isotorachrysone (3), asperflavin (7), emodin (9), physcion (10), questin (11), catenarin (12), rubrocristin (13), and physcion bianthrones (15)]. In this paper, we describe the isolation, structure, stereochemistry, and cytotoxicity against the HL-60 and A-549 cell lines of the new compounds.

Results and Discussion Compound 2 was a new analogue of aspergiolide A (1), and both compounds had similar UV spectra [λmax (log ε) 202 (4.36), 233 (4.31), 308 (4.01), 434 (3.58) for 2; λmax (log ε) 203 (4.25), 235 (4.19), 307 (3.90), 431 (3.61) nm for 17]. The HRESIMS of 2 (m/z 473.0868 [M - H]-, calcd 473.0873) established the molecular formula as C26H18O9. The 1H and 13C NMR data of 2 (Table 1) were similar to those of 18 except that the signals of a phenolic hydroxy group in the structure of 1 were replaced by those of a methoxy group (δH 3.88, δC 56.1). The HMBC spectrum also supported the existence of the naphtho[1,2,3-de]chromene-2,7-dione skeleton (Figure 1), and the methoxy group was located at C-8 via the correlation between the δH 3.88 and δC 164.1 resonances. Therefore, compound 2 was identified as the 8-methoxy derivative of 1 and was named aspergiolide B (2). Compound 4 was obtained as yellow needles. Its molecular formula C19H22O8 was determined by HRESIMS (m/z 377.1254 [M - H]-, calcd 377.1236). Comparison of the 1H and 13C NMR * To whom correspondence should be addressed. Tel: 0086-53282032065. Fax: 0086-532-82033054. E-mail: [email protected].

10.1021/np800303t CCC: $40.75

Table 1. 1H and 13C NMR and HMBC Data for Compound 2a position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 8-OCH3 6-OH 12-OH 20-OH 22-OH

δH (J/Hz)

7.00, s 6.75, s

7.20, s

2.45, s

6.09, s 6.24, d (2.2) 2.54, s 3.88, s 11.16, s 13.84, s 10.50 br, s 10.35, s

HMBC (HfC)

δC 159.1, qC 131.7, qCb 132.5, qC 135.6, qCb 109.8, CH 163.4, qC 102.5, CH 164.1, qC 111.7, qC 184.6, qCc 109.2, qC 159.2, qC 122.0, CH 134.3, qC 141.6, qC 115.6, qC 15.8, CH3 191.2, qCc 115.4, qC 162.6, qC 100.7, CH 162.6, qC 111.8, CH 145.1, qC 23.1, CH3 56.1, CH3

3, 6, 7, 9 5, 6, 8, 9

11, 12, 15, 17

13, 14, 15

19, 20, 22, 23 19, 21, 22, 25 19, 23, 24 8 5, 6, 7 11, 12, 13

a Spectra were recorded in DMSO-d6 at 600 MHz for 1H NMR and HMBC, and 150 MHz for 13C NMR using TMS as internal standard. b,cSignals can be interchanged.

Figure 1. Selected HMBC correlations of compounds 2 and 4. data (Table 2) with those of isotorachrysone (3)9 suggested that they had the same naphthalene skeleton (Figure 1). Further comparison of their 1D NMR spectra indicated that compound 4

 2008 American Chemical Society and American Society of Pharmacognosy Published on Web 11/06/2008

1838 Journal of Natural Products, 2008, Vol. 71, No. 11

Du et al.

Table 2. 1H and 13C NMR Data for Compounds 3-6a 3b position 1 2 3 4 5 6 7 8 9 10 6-OH 1-OH 1′ 2′ 3′ 4′ 1′′ 2′′ 3′′ 4′′ 5′′ 2′′-OH 3′′-OH 5′′-OH

δH (J/Hz)

6.89, s 6.66, d (2.2) 6.57, s

4c δC 154.0, qC 122.9, qC 135.3, qC 119.0, CH 102.9, CH 159.0, qC 98.4, CH 157.8, qC 108.4, qC 138.8, qC

8.84, s 9.75, s 2.49, s 2.24, s 4.11, s

δH (J/Hz)

7.01, s 6.90, d (1.8) 6.67, d (1.8)

5c δC

δH (J/Hz)

152.0, qC 122.7, qC 133.9, qC 118.8, CH 102.6, CH 156.6, qC 98.6, CH 157.3, qC 108.6, qC 136.8, qC

6.47, s 6.95, s 6.86, d (1.8) 6.58, d (1.8)

9.73, s 204.0, qC 32.3, CH3 20.2, CH3 56.8, CH3

2.52, s 2.22, s 4.01, s 5.73, d (4.8) 4.14, m 3.98, m 4.04, m 3.52, m 4.75, d (8.7) 4.96, d (5.5) 4.86, t (5.5)

204.3, qC 32.1, CH3 19.6, CH3 56.4, CH3 100.2, CH 71.6, CH 69.4, CH 86.5, CH 61.6, CH2

a Spectra were recorded at 600 MHz for 1H NMR and 150 MHz for in DMSO-d6.

13C

was a furanoside. The connection between the sugar and naphthalene moieties through the O bond was established by the key HMBC correlation from H-1′′ (δH 5.73) to C-6 (δC 156.6). The ribose residue was confirmed through comparing the 13C NMR data with those of several furanosides, such as methyl ribofuranosides10 and asperflavin ribofuranoside.11 D-Ribose in 4 was established by measurement of its optical rotation following acid hydrolysis ([R]20D -18.5, c 0.085, H2O).12 The sugar moiety was further determined as R-D-ribofuranose by comparison of the J1′′,2′′ value (4.8 Hz) with those of the methyl-R-D-ribofuranoside (J1,2 ) 4.3 Hz) and methylβ-D-ribofuranoside (J1,2 ) 1.2 Hz).13 Thus, compound 4 was identified as isotorachrysone-6-O-R-D-ribofuranoside.

6c δC 153.7, qC 110.1, CH 137.4, qC 117.2, CH 102.6, CH 155.4, qC 97.8, CH 156.9, qC 108.9, qC 137.0, qC

9.12, s 2.31, s 3.97, s 5.67, d (4.8) 4.09, m 3.94, m 3.99, m 3.48, m 4.70, d (9.1) 4.93, d (5.5) 4.83, t (5.5)

δH (J/Hz) 6.62, dd (7.8, 0.9) 7.25, t (8.2, 7.8) 7.16 br, d (7.3) 6.96 d (2.2) 6.66, d (2.2)

δC 154.1, qC 108.3, CH 127.9, CH 117.7, CH 103.0, CH 155.2, qC 98.6, CH 156.9, qC 110.7, qC 136.9, qC

9.22, s 3.99, s 21.2, CH3 56.2, CH3 100.2, CH 71.5, CH 69.3, CH 86.3, CH 61.5, CH2

5.70, d (4.8) 4.10, m 3.95, m 4.00, m 3.49, m 4.70, d (9.1) 4.93, d (5.5) 4.83, t (5.5)

56.3, CH3 100.2, CH 71.5, CH 69.3, CH 86.4, CH 61.5, CH2

NMR using TMS as internal standard. b Measured in acetone-d6. c Measured

Compounds 5 and 6 were both analogues of 4, and their molecular formulas were established by HRESIMS as C17H20O7 and C16H18O7, respectively. Careful comparison of their 1H and 13C NMR spectra with those of 4 revealed that the structures contained the same sugar residue and the only substituent differences appeared on the naphthalene moiety. The resonances of the acetyl group in 4 were absent in 5, and a new meta-coupled aromatic proton resonance (δ 6.47) appeared at this position. The corresponding C-2 carbon shifted upfield from δ 122.7 to δ 110.1. In compound 6, both the resonances of the acetyl group and CH3-3′ were absent and two ortho-coupled aromatic proton signals (δ 6.62 and 7.25) appeared correspondingly at C-2 (δ 108.3) and C-3 (δ

Cytotoxic Polyketides from Aspergillus glaucus

Journal of Natural Products, 2008, Vol. 71, No. 11 1839

Table 3. 1H and 13C NMR Data for Compounds 7 and 8a 7 position 1 2 3 4 5 6 7 8 9 10 3-CH3 8-OCH3 4a 8a 9a 10a 3-OH 4-OH 6-OH 9-OH

δH (J/Hz) 2.82, d (16.9) 2.62, dd (16.9, 1.8)

8 δC

2.72, dd (4.6, 17.4) 2.51, dd (10.5, 17.4) 2.07, m

69.4, qC 2.96, d (16.0) 2.87, d (14.6) 6.54, d (2.2) 6.41, d (2.2) 6.79, s 1.26, s 3.84, s

δC

δH (J/Hz)

203.2, qC 51.4, CH2

42.7, CH2 101.9, CH 160.4, qC 97.8, CH 161.2, qC 165.0, qC 115.7, CH 28.9, CH3 55.7, CH3 137.8, qC 108.9, qC 108.1, qC 141.6, qC

4.30 br, t (7.2, 7.7) 6.62, d (2.2) 6.45, d (1.8) 7.10, d (0.8) 1.05, d (6.4) 3.85, s

202.5, qC 43.0, CH2 36.3, CH 71.9, CH 102.6, CH 160.6, qC 98.1, CH 161.2, qC 165.2, qC 113.8, CH 17.8, CH3 55.7, CH3 141.6, qC 108.3, qCb 108.0, qCb 142.2, qC

4.83, s 5.53, d (6.9) 10.34, s 15.10 (s)

10.28, s 14.97, s

a Spectra were recorded in DMSO-d6 at 600 MHz for 1H NMR and 150 MHz for interchanged.

127.9). Therefore, 5 and 6 were identified as 8-methoxy-3-methyl1-naphthalenol-6-O-R-D-ribofuranoside and 8-methoxy-1-naphthalenol-6-O-R-D-ribofuranoside, respectively. To date, less than 10 naphthyl furanosides have been identified as natural products, and only one naphthyl ribofuranoside has been reported previously.14,15 Compound 8 had the same molecular formula, C16H16O5, established by the HRESIMS (m/z 287.0912 [M - H]-, calcd 287.0919), as the known pigment asperflavin (7).16,17 Careful comparison of their 1D NMR data (Table 3) indicated that they had the same skeleton and only OH-3 in 7 shifted to C-4 in 8. This was confirmed by analyzing the peak shapes and J values of H-2 (δ 2.72, dd, J ) 4.6, 17.4; δ 2.51, dd, J ) 10.5, 17.4), H-3 (δ 2.07, m), and H-4 (δ 4.30, brt, J ) 7.2, 7.7). The J3,4 (7.7 Hz) value helped to establish the trans configuration of C-3/C-4.18 The absolute configuration was thus determined to be 3R, 4S by the maximal negative Cotton effect at 281.0 nm and the maximal positive effect at 223.4 nm in the CD spectrum.19 Therefore, the structure of 8 was established, and the compound was named isoasperflavin. Compound 14 was a yellow pigment with the molecular formula C20H18O9 established by HRESIMS (m/z 403.1021 [M + H]+, calcd 403.1029). Both the UV spectra [λmax (log ε) 440 (3.45), 286 (3.88), 251 (3.75), 223 (4.05)] and the 1D NMR data (Table 4) were consistent with those of the known compound variecolorquinone A, which was isolated from the metabolites produced by the halotolerant fungal strain Aspergillus Variecolor B-17.20 Because they had opposite specific rotations ([R]20D ) +16.8 for 14; [R]20D ) -18.0 for variecolorquinone A20), the absolute configuration of C-3′′ in 14 was deduced as R and 14 was named (+)variecolorquinone A. The new bianthrone isomers (16 and17) had UV spectra (λmax 239, 278, and 360) characteristic of bianthrones.21 Their HRESIMS (m/z 523.1409 and 523.1400 [M - H]-, calcd 523.1393) established the same molecular formula of C31H24O8. According to their 1H NMR spectra (Table 4), they both had eight meta-coupled aromatic protons, two methyl groups, five phenolic hydroxy groups, and one methoxy group. Comparison of their 1D NMR data (Table 4) with those of the physcion bianthrones (15),22 emodin bianthrones,23,24 and the anthracene monomers physcion and emodin25 indicated they were two C-10/C-10′ isomers of physcion-emodin bianthrones. Furthermore, in the 1H NMR spectra of 16 and 17, the chemical shifts of OH-1/1′ were more upfield and the shifts of OH-6 and

13C

NMR using TMS as internal standard.

b

Signals can be

OH-8/8′ were more downfield in 16 than in 17. Comparison with the literature data indicated that 16 and 17 respectively had a trans and a cis relationship between H-10/H-10′.21,23,24 Aromatic polyketides differ from other polyketides by their characteristic polycyclic aromatic structures.26 They are produced by repetitive Claisen condensations of a starter unit (a dedicated FAS, another PKS or an acyl CoA) with malonyl-CoA elongation units. The biosynthesis of polyketides in fungi is governed by iterative type I polyketide synthases (PKS), which are multifunctional proteins consisting of domains for individual enzyme activities.1,2,26 Aspergiolide B (2) may have a biosynthetic pathway similar to aspergiolide A (1), probably using the same PKS but with a different anthraquinone precursor, rubrocristin (13).7 Compound 2 may also be formed through postmodification of compound 1, such as O-methylation by S-adenosylmethionine (Figure 2, A). The biogenetic relationships of the other polyketides (3-17) isolated herein are postulated (Figure 2, B). With the accumulation of knowledge about the enzymology and the genome of A. glaucus, investigation strategies employing precursor-directed biosynthesis27 and mutational biosynthesis28 become feasible for further studies on aspergiolide polyketides. The new compounds were evaluated for their cytotoxicities against the HL-60 cell line by the MTT method29 and the A-549 cell line by the SRB method.30 Aspergiolide B (2) showed potent cytotoxicities against the HL-60 and A-549 cell lines with IC50 values of 0.51 and 0.24 µM, respectively, indicating that the O-methylation of OH-8 did not negatively impact its activities.7 Compound 16, (trans)-emodin-physcion bianthrone, showed moderate cytotoxicities against the HL-60 and A-549 cell lines with IC50 values of 7.8 and 9.2 µM, respectively. Its cis-isomer, compound 17, showed comparable cytotoxicities, and the IC50 values were 44.0 and 14.2 µM, respectively. It appears that the isomerization of the C-10/C-10′ axis does not impact the activities of the emodinphyscion bianthrones. The other new compounds showed no cytotoxicity at 100 µM against either cell line, suggesting that the naphtho[1,2,3-de]chromene-2,7-dione skeleton may be important for cytotoxicity and warrants further investigation to identify the molecular targets for the aspergiolides. Experimental Section General Experimental Procedures. Melting points were measured using a Yanaco MP-500D micromelting point apparatus and were

position

5.06 br, s 4.76 br, s

4.22, dd (6.4, 10.9) 4.37, dd (4.1, 10.9) 3.78, m 3.42, m 13.65, s

66.8, CH2 69.2, CH 62.5, CH2

55.9, CH3 166.0, qC

3.89, s

12.19, s/12.14, s

11.83, s/11.89, s

55.6, CH3/55.6, CH3

3.84, s/3.82, s

110.8, qC/110.9, qC

109.9, qC 22.0, CH3/22.0, CH3

142.8, qC/143.2, qC

136.4, qC

2.29, s/2.31, s

140.1, qC/140.5, qC

132.2, qC

19.4, CH3

114.2, qC/114.3, qC

115.0, qC

56.5, CH/56.5, CH

182.5, qC

4.36, s/4.34, s

190.3, qC/190.3, qC

184.7, qC

100.1, CH/100.1, CH

165.1, qC/165.2, qC

107.8, CH/107.7, CH

120.8, CH/120.7, CH

164.5, qC/164.6, qC

6.01 br, s/5.97 br, s

6.38, d (2.3)/6.35, d (2.3)

6.68, s/6.70, s

146.8, qC/146.7, qC

164.1, qC

105.2, CH

2.36, s

6.78, d (2.2)

109.6, CH

7.15, d (2.2)

169.0, qC

118.7, CH

142.1, qC

116.9, CH/117.0, CH

6.10, s/6.12, s

δC

128.7, CH

δH (J/Hz) 161.9, qC/161.8, qC

δC

158.5, qC

7.45 br, s

δH (J/Hz)

15c,f,g

11.75, s 11.70, s 9.79, s 12.05, s 12.02, s

3.89, s

2.23, s/2.24, sg

4.56, s 4.56, s

6.10 br, s 6.14 br, s

6.62, s 6.62, s 6.31 br, s 6.40, d (2.2)

6.38 br, s 6.31 br, s

δH (J/Hz)

16d δC

δC

2.27, s/ 2.29, sg

55.3, CH3

11.82, s 11.79, s 9.75, s 11.97, s 11.97, s

3.84, s

56.1, CH3

21.8, CH3/21.9, CH3f

111.0, qC/111.5, qCf

110.3, qC/110.9, qCf 20.9, CH3 20.9, CH3

144.2, qC/144.7, qCf

141.7, qC/142.0, qCf

115.0, qC/115.1, qCf 140.2, qC 140.2, qC 144.1, qC/144.4, qCf

114.0, qC/113.9, qCf

56.7, CH/56.8, CHf

55.8, CH/55.9, CHf

109.9, CH 108.4, CH 166.3, qC 167.8, qC 102.6, CH 101.2, CH 165.2, qC/165.4, qCf

122.0, CH/122.1, CHf

162.7, qC 162.7, qC 117.2, CH 117.2, CH 147.5, qC/147.8, qCf

191.1, qC/191.4, qCf 4.56, s 4.56, s

6.15 br, s 6.18 br, s

6.66, s 6.69, s 6.26, d (2.3) 6.34, d (2.3)

6.34 br, s 6.27 br, s

δH (J/Hz)

190.2, qC/190.4, qCf

121.1, CH 121.1, CH 108.9, CH 107.5, CH 165.7, qC nde 101.7, CH 100.3, CH 164.3, qC/164.5, qCf

161.7, qC 161.7, qC 116.4, CH 116.4, CH 146.5, qC/146.6, qCf

17d

a Spectra were recorded in DMSO-d6 at 600 MHz for 1H NMR and 150 MHz for 13C NMR using TMS as internal standard. b Measured in DMSO-d6. c Measured in CDCl3. d Measured in acetone-d6. e Signals not detected. f Signals for Cx and Cx′ (x ) 1, 2, 3,...) can be interchanged. g Signals for Hx and Hx′ (x ) 1, 2, 3,...) can be interchanged.

3′′ 4′′ 1-OH 1′-OH 6-OH 8-OH 8′-OH 3′′-OH 4′′-OH

1 1′ 2 2′ 3 3′ 4 4′ 5 5′ 6 6′ 7 7′ 8 8′ 9 9′ 10 10′ 1a 1a′ 4a 4a′ 5a 5a′ 8a 8a′ 3-CH3 3′-CH3 6-OCH3 6′-OCH3 8-OCH3 1′′ 2′′

14b

Table 4. 1H and 13C NMR Data for Compounds 14-17a

1840 Journal of Natural Products, 2008, Vol. 71, No. 11 Du et al.

Cytotoxic Polyketides from Aspergillus glaucus

Journal of Natural Products, 2008, Vol. 71, No. 11 1841

Figure 2. Postulated biosynthetic pathways for aspergiolide B (2, A) and polyketides 3-17 (B). uncorrected. Specific rotations were obtained on a Jasco P-1020 digital polarimeter. UV spectra were recorded on Beckman DU 640 spectrophotometer. IR spectra were taken on a Nicolet Nexus 470 spectrophotometer in KBr discs. 1H NMR, 13C NMR, and DEPT spectra and 2D-NMR were recorded on a JEOL JNM-ECP 600 spectrometer using TMS as internal standard, and chemical shifts were recorded as δ values. ESIMS were measured on a Q-TOF Ultima Global GAA076 LC mass spectrometer. Semiprepartive HPLC was performed using an ODS column [YMC-pak ODS-A, 10 × 250 mm, 5 µm, 4 mL/min]. Fungal Material. The fungus Aspergillus glaucus was obtained from marine sediment surrounding mangrove roots collected in the Fujian Province, China. It was identified by Prof. Li Tian, the First Institute of Oceanography, SOA, Qingdao, China, and preserved in the China Center for Type Culture Collection (patent depositary number CCTCC M 206022). Working stocks were prepared on potato dextrose agar slants stored at 4 °C. Fermentation and Extraction. Spores were directly inoculated into 500 mL Erlenmeyer flasks containing 100 mL fermentation media (mannitol 20 g, maltose 20 g, glucose 10 g, monosodium glutamate 10 g, KH2PO4 0.5 g, MgSO4 · 7H2O 0.3 g, yeast extract 3 g, and corn steep liquor 1 g, dissolved in 1 L of seawater, pH 6.5). The flasks were incubated on a rotatory shaker at 165 rpm at 28 °C. After 9 days of cultivation, 15 L of whole broth was filtered through cheesecloth to separate the broth supernatant and mycelia. The former was extracted with EtOAc, while the latter was extracted with acetone. The acetone extract was evaporated under reduced pressure to afford an aqueous solution and then extracted with EtOAc. The two EtOAc extracts were combined and concentrated in vacuo to give a crude gum (30 g). Purification of the New Compounds. The crude gum (30 g) was subjected to silica gel column chromatography (CHCl3/MeOH, v/v, gradient), and the active fractions 3, 4, 5, and 6 eluted with the solvent

CHCl3/MeOH (40:1, 20:1, 10:1, and 9:1) were separately subjected to repeated Sephadex LH-20 column chromatography (CHCl3/MeOH, 1:1). The active subfractions 5-5-2, 6-2-2, 5-5-1-4, and 4-6-6-1 were further purified respectively by HPLC using a reversed-phase C18 column (50% MeOH, 30% CH3CN, 35% CH3CN, and 75% CH3CN, 4.0 mL/min), to give compounds 4 (21.3 mg), 5 (2.8 mg), 6 (4.5 mg), 8 (3.0 mg), 16 (3.2 mg), and 17 (4.1 mg). Another two subfractions, 5-5-2-5 and 6-34, were respectively subjected to repeated Sephadex LH-20 column chromatography (MeOH) to give compounds 2 (10 mg) and 14 (24 mg). Acid Hydrolysis of 4 and Determination of the Configuration of the Ribofuranose. A solution of 4 (9 mg) in 6 mol/L HCl (1 mL) was reacted for 3 h at 100 °C. The reaction mixture was extracted with EtOAc repeatedly to remove the aglycone fraction, which was identical to isotorachrysone (3).7 The H2O layer was then concentrated to furnish the sugar residue (1.7 mg). The rotation recorded for the ribose isolated in this study was [R]20D -18.5 (c 0.085, H2O), which closely matched that for the D-ribose (lit. -20).9 Biological Assays. Cytotoxic activity was evaluated using the HL60 cell line by the MTT method29 and the A-549 cell line by the SRB method.30 In the MTT assay, the cell line was grown in RPMI-1640 supplemented with 10% FBS under a humidified atmosphere of 5% CO2 and 95% air at 37 °C. Cell suspensions (200 µL) at a density of 5 × 104 cells/mL were plated in 96-well microtiter plates and incubated for 24 h. The test compound solutions (2 µL in MeOH) at different concentrations were added to each well and further incubated for 72 h under the same conditions. MTT solution (20 µL of a 5 mg/mL solution in IPMI-1640 medium) was added to each well and incubated for 4 h. Old medium (150 µL) containing MTT was then gently replaced by

1842 Journal of Natural Products, 2008, Vol. 71, No. 11 DMSO and pipetted to dissolve any formazan crystals formed. Absorbance was then determined on a Spectra Max Plus plate reader at 540 nm. In the SRB assay, cell suspensions (200 µL) were plated in 96-cell plates at a density of 2 × 105 cells/mL. Then the test compound solutions (2 µL in MeOH) at different concentrations were added to each well and further incubated for 24 h. Following drug exposure, the cells were fixed with 12% trichloroacetic acid and the cell layer was stained with 0.4% SRB. The absorbance of SRB solution was measured at 515 nm. Dose-response curves were generated, and the IC50 values were calculated from the linear portion of log dose response curves. Aspergiolide B (2): red solid (MeOH); UV (MeOH) λmax (log ε) 202 (4.36), 233 (4.31), 308 (4.01), 434 (3.58); IR (KBr) νmax cm-1 3133, 2925, 1678, 1588, 1439, 1377, 1337, 1236, 1204, 1173, 1083 cm-1; 1H and 13C NMR, see Table 1; HRESIMS m/z 473.0868 [M H]- (calcd for C26H17O9, 473.0873). Isotorachrysone-6-O-r-D-ribofuranoside (4): yellow needles (MeOH); mp 174-176 °C; [R]20D +178.6 (c 0.11, MeOH); UV (MeOH) λmax (log ε) 245 (3.41), 308 (3.33), 337 (3.29); IR (KBr) νmax 3390, 3289, 2966, 2924, 2850, 1642, 1619, 1576, 1456, 1394, 1258, 1157, 1106, 1017, 803 cm-1; 1H and 13C NMR, see Table 2; HRESIMS m/z 377.1254 [M - H]- (calcd for C19H21O8, 377.1236). 8-Methoxy-3-methyl-1-naphthalenol-6-O-r-D-ribofuranoside (5): yellow needles (MeOH); mp 176-178 °C; [R]20D +110.7 (c 0.09, MeOH); UV (MeOH) λmax (log ε) 237 (4.00), 303 (3.43), 318 (3.36), 333 (3.35); IR (KBr) νmax 3409, 2962, 2927, 2853, 1638, 1614, 1583, 1455, 1385, 1362, 1253, 1164, 1102, 1012, 849 cm-1; 1H and 13C NMR, see Table 2; HRESIMS m/z 359.1118 [M + Na]+ (calcd for C17H20O7Na, 359.1107). 8-Methoxy-1-naphthalenol-6-O-r-D-ribofuranoside (6): yellow oil (MeOH); [R]20D +137.7 (c 0.15, MeOH); UV (MeOH) λmax (log ε) 244 (3.81), 299 (3.63), 321 (3.51), 335 (3.52); IR (KBr) νmax 3402, 2926, 1633, 1618, 1591, 1454, 1402, 1376, 1317, 1252, 1166, 1124, 1042, 1003 cm-1; 1H and 13C NMR, see Table 2; HRESIMS m/z 345.0943 [M + Na]+ (calcd for C16H18O7Na, 345.0950). Isoasperflavin (8): yellow solid (MeOH); [R]20D +34.1 (c 0.075, MeOH); CD (MeOH) λmax (∆ε) 223.4 (0.9), 281.0 (-0.5); UV (MeOH) λmax (log ε) 233 (3.94), 268 (3.06), 318 (3.33), 331 (3.27), 392 (3.62); IR (KBr) νmax 3367, 2923, 2853, 1681, 1595, 1368, 1260, 1202, 1091, 1045 cm-1; 1H and 13C NMR, see Table 3; HRESIMS m/z 287.0912 [M - H]- (calcd for C16H15O5, 287.0919). (+)-Variecolorquinones A (14): yellow solid (MeOH); [R]25D +16.8 (c 0.03, MeOH); UV (CHCl3) λmax (log ε) 440 (3.45), 286 (3.88), 251 (3.75), 223(4.05); IR (KBr) νmax 3425, 1718, 1672, 1635, 1598, 1574 cm-1; 1H and 13C NMR, see Table 4; HRESIMS m/z 403.1021 [M + H]+ (calcd for C20H19O9, 403.1029). (trans)-Emodin-physcion bianthrone (16): yellow solid (MeOH); [R]20D -8.4 (c 0.10, CHCl3); UV (CHCl3) λmax (log ε) 239 (4.20), 278 (4.14), 360 (4.23); IR (KBr) νmax 3354, 2971, 2926, 2854, 1638, 1619, 1595, 1486, 1456, 1362, 1327, 1277, 1259, 1164, 1066 cm-1; 1H and 13 C NMR, see Table 4; HRESIMS m/z 523.1409 [M - H]- (calcd for C31H23O8, 523.1393). (cis)-Emodin-physcion bianthrone (17): yellow solid (MeOH); [R]20D +9.8 (c 0.06, CHCl3); UV (CHCl3) λmax (log ε) 239 (4.07), 278 (3.98), 360 (4.03);1H and 13C NMR, see Table 4; IR (KBr) νmax 3353, 2980, 2924, 2853, 1637, 1618, 1595, 1487, 1457, 1361, 1329, 1259, 1189, 1160, 1065 cm-1; HRESIMS m/z 523.1400 [M - H]- (calcd for C31H23O8, 523.1393).

Du et al. Acknowledgment. This work was financially supported by the Chinese National Science Fund (No. 30672527) and the Shandong Provincial Natural Science Fund (No. Z2006C13). The antitumor assays were performed at the Shanghai Institute of Materia Medica, Chinese Academy of Sciences. References and Notes (1) Julia, S.; Christian, H. J. Biotechnol. 2006, 124, 690–703. (2) Cox, R. J. Org. Biomol. Chem. 2007, 5, 2010–2026. (3) Ashley, J. N.; Raistrick, H.; Richards, T. Biochem. J. 1939, 33, 1291– 1303. (4) Heidrun, A.; Ilham, K.; Hans, Z.; Hartmut, L. Arch. Microbiol. 1980, 126, 223–230. (5) Bachmann, M.; Luthy, J.; Schlatter, C. J. Agric. Food Chem. 1979, 27, 1342–1347. (6) George, B.; Dieter, H. K.; Shank, R. C.; Steven, M. W.; Wogan, G. N. J. Org. Chem. 1971, 36, 1143–1147. (7) Du, L.; Zhu, T.; Fang, Y.; Liu, H.; Gu, Q.; Zhu, W. Tetrahedron 2007, 63, 1085–1088. (8) Du, L.; Zhu, T.; Fang, Y.; Liu, H.; Gu, Q.; Zhu, W. Tetrahedron 2008, 64, 4657. (9) Lin, C. N.; Lu, C. M.; Lin, H. C.; Ko, F. N.; Teng, C. M. J. Nat. Prod. 1995, 58, 1934–1940. (10) Thomas, E. W.; Harry, P. C. H.; Terry, E. N.; Nick, A. M. Biochemistry 1974, 13, 2650–2655. (11) Li, Y.; Li, X.; Lee, U.; Kang, J. S.; Choi, H. D.; Sona, B. W. Chem. Pharm. Bull. 2006, 54, 882–883. (12) Robert, K. N.; Harry, W. D.; Hewitt, G. F., Jr. J. Am. Chem. Soc. 1954, 76, 763–767. (13) Anthony, S. S.; Robert, B. J. Org. Chem. 1984, 49, 3292–3300. (14) Antkowiak, W. Z.; Krzyzosiak, W. J.; Biernat, J.; Ciesiolka, J.; Gornicki, P. Bull. Acad. Pol. Sci., Ser. Sci. Chim. 1977, 25, 173–178. (15) Antkowiak, W. Z.; Krzyzosiak, W. J. Bull. Acad. Pol. Sci., Ser. Sci. Chim. 1977, 25, 421–425. (16) Fujimoto, H.; Fujimaki, T.; Okuyama, E.; Yamazaki, M. Chem. Pharm. Bull. 1999, 47, 1426–1432. (17) John, F. G. J. Chem. Soc., Perkin Trans. 1 1972, 2406–2411. (18) Bringmann, G.; Mu¨nchbach, M.; Messer, K.; Koppler, D.; Michel, M.; Schupp, O.; Wenzel, M.; Louis, A. M. Phytochemistry 1999, 51, 693–699. (19) Bringmann, G.; Messer, K.; Saeb, W.; Peters, E. M.; Peters, K. Phytochemistry 2001, 56, 387–391. (20) Wang, W.; Zhu, T.; Tao, H.; Lu, Z.; Fang, Y.; Gu, Q.; Zhu, W. J. Antibiot. 2007, 60, 603–607. (21) Politi, M.; Sanogo, R.; Ndjoko, K.; Guilet, D.; Wolfender, J. L.; Hostettmann, K.; Morelli, I. Phytochem. Anal. 2004, 15, 355–364. (22) Monache, G. D.; Rosa, M. C.; Scurria, R.; Monacelli, B.; Pasqua, G.; Olio, G. D.; Botta, B. Phytochemistry 1991, 30, 1849–1854. (23) Mai, L. P.; Gueritte, F.; Dumontet, V.; Tri, M. V.; Hill, B.; Thoison, O.; Guenard, D.; Sevenet, T. J. Nat. Prod. 2001, 64, 1162–1168. (24) Donald, W. C.; John, S. E.; Warwick, D. R. Aust. J. Chem. 1976, 29, 1535–1548. (25) Danielsen, K.; Aksnes, D. W. Magn. Reson. Chem. 1992, 30, 359– 360. (26) Shen, B. Top. Curr. Chem. 2000, 209, 1–51. (27) Thiericke, R.; Rohr, J. Nat. Prod. Rep. 1993, 10, 265–289. (28) Weist, S.; Su¨ssmuth, R. D. Appl. Microbiol. Biotechnol. 2005, 68, 141–150. (29) Mosmann, T. J. Immunol. Methods 1983, 65, 55–63. (30) Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J.; Vistica, D.; Warren, J. T.; Bokesch, H.; Kenney, S.; Boyd, M. R. J. Natl. Cancer Inst. 1990, 82, 1107–1112.

NP800303T